Complicated patterns of gene regulation underlie development of higher organisms, and many known diseases are associated with aberrant gene regulation. The proposed experiments are designed to discover fundamental mechanisms of gene regulation. To this end studies are proposed primarily with yeast and mammalian cells; the former emphasize the use of genetics in combination with biochemistry, and the use of disparate organisms allows the generality of any given discovery to be tested. It is proposed to create and study transcriptional activators that bypass the ordinary requirements for transcriptional activating regions. It will be tested whether these "non-classical" polymerase activators, which work extremely powerfully in yeast, do so by forming part of the RNA polymerase II holoenzyme and recruit that structure to DNA. These experiments will be paralleled by experiments with classical activators that will attempt to identify targets of transcriptional activating regions and to determine whether activating region-target interactions trigger gene activation by holoenzyme recruitment. Further experiments with both classes of activators will be used to distinguish between models for how specific repressors work. The role of nucleosomes in helping and hindering gene activation will be analyzed using these two classes of activators; and the mechanism of overcoming the inhibitory effect of nucleosomes will be explored.